Design and synthesis of fluorescent sensors for zinc ion derived from 2-aminobenzamide

Article abstract of DOI:10.1002/ejoc.201100388

Four novel Zn²⁺ fluorescent sensors have been designed with N, N-bis(2-pyridylmethyl)ethylenediamine as chelator and 2-aminobenzamide as fluorophore. These sensors were prepared in two or three steps from readily available starting materials. Of the four designed sensors, ZnABA was found to be the most efficient Zn²⁺-specific fluorescent probe and has good solubility in biological buffer, a large Stokes shift (186 nm), a high off-on fluorescence response (16-fold enhancement), and distinct selectivity towards Zn²⁺ over other metal ions. Our results have demonstrated an excellent linear relationship between the fluorescence intensity of ZnABA and the Zn²⁺ concentration from 0 to 10 μM, which indicates that ZnABA has the potential to be used for the quantitative determination of Zn ²⁺ in an aqueous environment.

Full text of DOI:10.1002/ejoc.201100388

FULL PAPER
DOI: 10.1002/ejoc.201100388
Design and Synthesis of Fluorescent Sensors for Zinc Ion Derived from
2-Aminobenzamide
Jia Jia,[a] Zhen-yuan Gu,[a] Ri-chen Li,[a] Ming-hao Huang,[a] Cai-shuang Xu,[a]
Yi-fei Wang,[b] Guo-wen Xing,*[a] and Yun-sheng Huang*[c]
Keywords: Sensors / Fluorescent probes / Fluorescence / Zinc
Four novel Zn2+ fluorescent sensors have been designed with
N,N-bis(2-pyridylmethyl)ethylenediamine as chelator and 2-
(186 nm), a high off–on fluorescence response (16-fold en-
hancement), and distinct selectivity towards Zn2+ over other
aminobenzamide as fluorophore. These sensors were pre- metal ions. Our results have demonstrated an excellent linear
pared in two or three steps from readily available starting
materials. Of the four designed sensors, ZnABA was found
to be the most efficient Zn2+-specific fluorescent probe and
has good solubility in biological buffer, a large Stokes shift
relationship between the fluorescence intensity of ZnABA
and the Zn2+ concentration from 0 to 10 μ
M, which indicates
that ZnABA has the potential to be used for the quantitative
determination of Zn2+ in an aqueous environment.
(BPEA),[10] N,N,NЈ-tris(2-pyridylmethyl)ethane-1,2-diamine
(TPEA),[11] and 1,4,7,10-tetraazacyclodecane.[12] Various
fluorophore structures have been reported, for example,
quinoline,[13] fluorescein,[9a,14] anthracene,[12a,15] benzoxaz-
ole,[11b,16] phthalimide,[17] and cyanine.[18] Although great
progress has been made in the synthesis of novel Zn2+ sen-
sors, the fluorophores used are essentially known fluores-
cent organic molecules. To the best of our knowledge, there
have been no reports of bioactive small molecules with fluo-
rescent properties being used for the design of Zn2+ sensors.
The development of novel multifunctional organic mole-
cules for Zn2+-specific fluorescent probes remains an inter-
esting and important challenge.
In recent years, novel 2-aminobenzamide analogues 1a–
1c (Figure 1) have been developed as heat shock protein 90
(Hsp90) inhibitors[19] with 1b being in multiple phase I clin-
ical trials.[19a] Previously, we reported[20] the molecular
mechanism of apoptosis of human chronic myeloid leuke-
mia (CML) K562 cells by 1b and developed a sensitive and
specific reversed-phase high-performance liquid chromatog-
raphy method for the identification and quantification of
1b in rat plasma. More recently, 1c (BJ-B11) was synthe-
Introduction
The zinc ion (Zn2+) is considered the second most abun-
dant heavy metal in the human body.[1] It has been found
that Zn2+ is widely distributed in all organisms, especially
in the brain,[2] pancreas,[3] and spermatozoa.[4] Many bio-
logical studies have implicated the significance of Zn2+ in
gene expression, apoptosis, enzyme regulation, and immu-
nological modulation, etc.[5] Zinc deficiency usually leads to
several diseases and disorders including anorexia, diabetes,
hypogonadism, immune dysfunction, and growth retar-
dation.[6] Therefore considerable effort has been focused on
developing highly sensitive and selective fluorescent sensors
to detect Zn2+ in biological and environmental systems.
In 1987, Frederickson et al.[7] reported the first Zn2+
-
specific fluorescent probe based on p-toluenesulfonamide–
quinoline. Over the years, many kinds of fluorescent sensor
molecules have been designed and synthesized for measur-
ing Zn2+ in vivo or in vitro.[8] In general, the chemical struc-
ture of a molecular probe for Zn2+ is composed of two com-
ponents: A chelator and a fluorophore. The most typical
chelators for Zn2+ include N,N-bis(2-pyridylmethyl)-
amine (BPA),[9] N,N-bis(2-pyridylmethyl)ethylenediamine
[a] Department of Chemistry, Beijing Normal University,
Beijing 100875, China
Fax: +86-10-58802075
E-mail: gwxing@bnu.edu.cn
[b] Guangzhou Jinan Biomedicine Research and Development
Center, National Engineering Research Center of Genetic
Medicine, Jinan University,
No. 601, West Huangpu Road, Guangzhou 510632, China
[c] School of Pharmacy, Guangdong Medical College,
Dongguan 523808, China
Fax: 86-769-22896547
E-mail: yshuang@gdmc.edu.cn
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100388.
Figure 1. Structures of 2-aminobenzamide analogues 1a–1c.
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G.-w. Xing, Y.-s. Huang et al.
FULL PAPER
sized and evaluated for in vitro antiviral activity against sev- influence of the indazolone substituent on the fluorescence,
eral viruses;[21] we found 1c to be potent against HSV, and di-indazolone–2-aminobenzamide 4 and 5 with no indaz-
it is currently being evaluated for use in anti-HSV clinical olone substituent were synthesized (Figure 2).
trials.
During the synthesis of these 2-aminobenzamides, it
came to our notice that compounds 1a–1c possess strong
Synthesis of the Zn2+ Sensors
fluorescent properties. As a part of our efforts to develop
The synthetic routes to ZnABA and 3–5 are shown in
new 2-aminobenzamide analogues and study their new
Scheme 1. All of these compounds can be easily prepared in
functions, herein, we report the design and synthesis of
two to three steps with readily available starting materials.
novel fluorescent sensors for Zn2+ based on 2-aminobenz-
amide.
Results and Discussion
Design of Zn2+ Sensors
A desirable Zn2+ sensor for biological and environmental
applications should possess unique characteristics and
properties, such as sufficient water solubility, high selectiv-
ity/sensitivity, long shelf-life, good photostability, and facile
preparation. Initially we screened the fluorescence response
of the known Hsp90 inhibitor 1a to different metal ions.
Unfortunately, no obvious fluorescence turn-on or quench-
ing was observed for any of the metal ions used. To develop
an efficient Zn2+ fluorescent probe, it was necessary to
modify the chemical structure of 1a.
Pyridyl-containing ligands such as BPA, BPEA, and
TPEA[9–11] have been reported to show high selectivity
towards Zn2+ over other metal ions. In this study, we intro-
duced BPEA as chelator for Zn2+ at the 2-position of 4-
indazolone-substituted benzamide. The chemical structure
of the first designed Zn2+ sensor (ZnABA, 2) is shown in
Figure 2. For comparison, the BPEA and indazolone moie-
ties on the benzamide were interchanged to make the sec-
ond Zn2+ sensor 3. Moreover, to further understand the
Scheme 1. Synthesis of 2-aminobenzamides. Reagents and condi-
tions: (a) K2CO3, DMF or NaH, DMF; (b) PdCl2, DPPF,
NaOtBu, toluene, 100 °C; (c) KOH, H2O2, EtOH/DMSO.
For the synthesis of ZnABA, initially, 2-bromo-4-fluoro-
benzonitrile (6) was coupled with indazol-4-one 7 in the
presence of NaH to afford the desired compound 8 in a
moderate yield (60%; Table 1, Entry 1). When the weaker
base K2CO3 was used instead of NaH for the reaction, a
more satisfactory yield (82%) was obtained (Table 1, En-
try 2), which indicates that the strong base NaH leads to
Table 1. Coupling of 7 with bromobenzonitrile by using different
bases.
Entry
Substrate
Base
Product
Yield [%]
1
2
3
4
5
6
6
NaH
K2CO3
NaH
K2CO3
NaH
8
60
82
40
92
n.d.
72
6
8
11
11
14
14
12
12
15
15
Figure 2. Structures of the newly designed Zn2+ sensors 2–5 with a
2-aminobenzamide scaffold.
K2CO3
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Fluorescent Sensors for Zinc Ion Derived from Aminobenzamide
many more complicated byproducts, as detected by TLC
during the substitution.
In addition, for Buchwald–Hartwig coupling reactions
with 12, 15, and 17 as substrates, higher coupling yields
Then BPEA was incorporated into the aromatic ring un- were achieved with lower substrate concentrations (Table 2,
der Buchwald–Hartwig conditions[22] by using catalytic Entries 3–8). These results are consistent with those ob-
PdCl2, 1,1Ј-bis(diphenylphosphanyl)ferrocene (DPPF), and tained with substrate 8.
NaOtBu in toluene at 100 °C. Note that the substrate con-
centration had a significant influence on the Pd-mediated
coupling yield (Table 2, Entries 1 and 2). The decrease in
the substrate (8) concentration from 0.05 to 0.025 m led to
Fluorescence Properties
a higher yield of the product (from 22 to 50%). This is due
With the synthesized Zn2+ sensors 2–5 in hand, the fluo-
to the fact that BPEA is poorly soluble in toluene. Thus,
rescence spectra were first recorded in 2-[4-(2-hydroxy-
the reaction should be more effective when performed at
ethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer
(pH = 7.4), and the results are shown in Figure 4. Com-
low concentrations.
pared with the strong fluorescence emissions of 1a and 5 at
Table 2. Effect of substrate concentration on the Buchwald–Hart-
around 440 nm, ZnABA, 3, and 4 exhibited weak fluores-
cence emissions, which showed their potential as off–on
Zn2+ sensors. However, upon addition of Zn2+ (1 equiv.),
only the emission intensity of ZnABA increased signifi-
cantly. Therefore, ZnABA was chosen as the Zn2+ sensor
suitable for further evaluation.
wig coupling reaction.
Entry Substrate
[Substrate] [molL–1]
Product Yield [%]
1
2
3
4
5
6
7
8
8
0.05
0.025
0.07
0.04
0.05
0.026
0.066
0.025
10
10
13
13
16
16
18
18
22
50
22
78
Ͻ5
44
33
59
8
12
12
15
15
17
17
Finally, hydration[23] of the resulting benzonitrile 10 with
KOH catalyzed by H2O2 in a mixture of EtOH/DMSO pro-
duced the Zn2+ sensor ZnABA (2) in high yield (87%). The
crystal structure of ZnABA was confirmed by single-crystal
X-ray diffraction (Figure 3).[24]
Figure 4. Fluorescence emission spectra of 1a and 2–5 in aqueous
solution (10 μm, 25 mm HEPES buffer, 0.1 m NaClO4, pH = 7.4, I
= 0.1). All the excitation wavelengths are at 260 nm.
Figure 3. X-ray crystallographic structure of ZnABA.
Table 3 shows the absorption and fluorescence properties
Similar synthetic methods were used to synthesize 2-ami- of all the 2-aminobenzamides used in this study. Compound
nobenzamides 3–5. It was observed that K2CO3 was a bet- 5 (without the indazolone moiety in its structure) has the
ter base for the coupling of indazol-4-one 7 with bromo- smallest ε value (1.35ϫ104 m–1 cm–1), whereas compound 4
benzonitriles 11 and 14 (Table 1, Entries 3–6). Notably, (with two indazolone moieties) possesses the highest ε value
when the aromatic nucleophilic substitution reaction of 4- (3.23ϫ104 m–1 cm–1) among the designed Zn2+ sensors (2–
bromo-2,6-difluorobenzonitrile (14) with 7 was carried out 5). ZnABA has the lowest fluorescence quantum yield (Φ0
with NaH as the base, the desired product was not obtained = 0.0096). After titration with 1 equiv. of Zn2+, the value
(Table 1, Entry 5), which suggests that 14 is very sensitive of Φ for the complex of ZnABA–Zn2+ increased signifi-
to strong bases and quickly decomposes under the reaction cantly (ΦZn = 0.077), thus showing an efficient off–on re-
conditions.
sponse with Zn2+
.
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Table 3. Absorption and fluorescence properties of 1a and 2–5.
demonstrate the formation of the ZnABA–Zn2+ complex
(Figure S1). Furthermore, a Job plot (Figure S2, Support-
ing Information) shows the 1:1 binding of ZnABA and
Zn2+. On the basis of all these results as well as the X-ray
crystallographic structure of free ZnABA, a Zn2+ binding
mode has been proposed (Figure S3).
[b]
Compound
ε[a] [m–1 cm–1]
λem [nm]
Φ[c]
1a
36500
28835
21900
27900
32380
13520
438
–
446
–
–
441
0.012
0.0096
0.077
0.014
0.015
0.286
2[d]
2+Zn2+[e]
3
4
5
The selectivity of ZnABA towards Zn2+ and other metal
ions was examined, and the results are shown in Figure 6.
No change in the fluorescence emission intensity was ob-
[a] Data were evaluated at the maximum λabs of 260 nm. [b] The
excitation wavelength was 260 nm. [c] The fluorescence quantum
yields were obtained by using quinine sulfate (in 0.05 m H2SO4, Φ
= 0.55) as the standard. [d] Data were determined in the absence
of Zn2+. [e] Data were determined in the presence of 1.0 equiv.
served upon addition of the metal cations Ba2+, Ca2+, Cr3+
,
Fe3+, Mg2+, Mn2+, Ni2+, and Pb2+, the transition-metal
ions Co2+ and Cu2+ quenched the fluorescence to some ex-
tent, and both Zn2+ and Cd2+ induced turn-on fluores-
cence, however, the fluorescence enhancement (16-fold) of
ZnABA towards Zn2+ is higher than that towards Cd2+ (8-
fold).
Zn2+
.
ZnABA readily dissolves in HEPES buffer (pH = 7.4)
and fluoresces weakly with λex and λem at 260 and 446 nm,
respectively. Addition of Zn2+ (0–1.5 equiv.) revealed that
the emission intensity is dependent upon the Zn2+ concen-
tration and reaches a maximum at Zn2+ Ն 1.0 equiv. (Fig-
ure 5). Its Stokes shift is 186 nm, which is much larger than
the reported values of many Zn2+ sensors.[9–18]
Figure 6. Selectivity of ZnABA towards Zn2+ and other metal ions.
Experimental conditions: ZnABA (10 μm; in 25 mm HEPES buffer,
0.1 m NaClO4, pH = 7.4, I = 0.1), 10 μm Ba2+, Ca2+, Co2+, Cr3+
,
Cu2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Cd2+, and Zn2+. λex
260 nm, λem = 446 nm.
=
For compounds 3 and 4, the BPEA and indazolone moi-
eties are interchanged on the aromatic ring with respect to
ZnABA. Although the electron-donating ability of the 2-
amino group decreased as a result of Zn2+ binding, the elec-
tron-withdrawing ability of the 1-carbamoyl group was not
Figure 5. Fluorescence emission spectra (excitation at 260 nm) of
ZnABA (10 μm; in 25 mm HEPES buffer, 0.1 m NaClO4, pH = 7.4,
I = 0.1) upon addition of Zn2+ [added as Zn(ClO4)2, 0–15 μm]. The
small peak at 520 nm arises from second-order scattering.
Note that the fluorescence emission intensity of ZnABA affected, because the 1-carbamoyl group cannot participate
was enhanced 16-fold upon addition of 1.0 equiv. of Zn2+ in the binding of Zn2+ along with BPEA. Therefore, the
.
This enhancement can be explained by the intermolecular fluorescence emission intensities of 3 and 4 upon addition
charge transfer (ICT) effect of the aromatic plane with of Zn2+ did not increase (Figures S4 and S5). For com-
BPEA as the conjugated electron donor and tetrahydroin- pound 5, there is no indazolone substituent in its scaffold.
dazol-4-one as the receptor. The UV titration spectra show It employs photoinduced electron transfer (PET) and exhib-
two absorption bands centered at 355 and 260 nm (Fig- its an on–off response with Co2+, Cu2+, Ni2+, and Zn2+
ure S1, Supporting Information). On addition of 0–1 equiv. (Figure S6).
Zn2+, the intensities of both bands decreased, and the origi-
As a Zn2+-specific fluorescent probe, the water-soluble
nal 355 nm band was blueshifted to 350 nm, which suggests ZnABA has the potential to be used for the quantitative
that on Zn2+ binding (1) the electron-donating ability of the determination of Zn2+ in an aqueous environment, because
2-amino group in the BPEA moiety is decreased and (2) the there is a good linear relationship between the fluorescence
electron-withdrawing ability of the 1-carbamoyl group is in- intensity of ZnABA and the concentration of Zn2+ between
creased. The clear isosbestic points at 317 and 275 nm also 0 and 10 μm (Figure S7).
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Fluorescent Sensors for Zinc Ion Derived from Aminobenzamide
washed with saturated sodium chloride solution (3ϫ 30 mL), and
dried with magnesium sulfate. After removal of the solvent, the
mixture was purified by column chromatography (hexanes/EtOAc,
4:1) and then recrystallized (CH2Cl2/hexanes) to give 8 (145 mg,
Conclusions
Four novel Zn2+ fluorescent sensors, ZnABA, 3, 4, and
5, have been designed with BPEA as the chelator and the
2-aminobenzamide scaffold as the fluorophore. Both the
use of the weaker base K2CO3 in the aromatic nucleophilic
substitution and a low substrate concentration in the Buch-
wald–Hartwig coupling reaction benefited the efficient
preparation of these probes. Of the four designed sensors,
ZnABA was found to be the most desirable Zn2+-specific
fluorescent probe and exhibited good solubility in bio-
logical buffer, a large Stokes shift (186 nm), a high off–on
fluorescence response (16-fold enhancement), and a distinct
selectivity towards Zn2+ over other metal ions. It has also
been demonstrated that ZnABA employs an ICT mecha-
nism, which is quite different from 5 for which PET oper-
ates, which suggests that indazolone substitution at the 4-
postion of the benzamide is necessary for ICT fluorescence.
Moreover, it is important that BPEA should be located at
the 2-position of the aromatic ring, which can efficiently
form a stable Zn2+ complex together with the 1-carbamoyl
group. In addition, the excellent linear relationship between
fluorescence emission intensity and Zn2+ concentration in-
dicates that ZnABA may be used in the quantitative deter-
mination of Zn2+ in aqueous systems. Owing to the special
properties of fluorescence and the pharmacological activity
of its core structure, it is hoped that ZnABA can be used
in biological applications.
1
0.41 mmol, 82%) as a colorless solid. Its H NMR spectrum was
consistent with that reported in ref.[19a]
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-4-(3,6,6-trimethyl-
4-oxo-4,5,6,7-tetrahydroindazol-1-yl)benzonitrile (10): A Schlenk
flask was charged with compound 8 (100 mg, 0.28 mmol,
1.00 equiv.), BPEA[26] (100 mg, 0.41 mmol, 1.50 equiv.), sodium
tert-butoxide (70 mg, 0.73 mmol, 2.60 equiv.), palladium chloride
(20 mg, 0.11 mmol, 0.40 equiv.), 1,1Ј-bis(diphenylphosphanyl)fer-
rocene (DPPF; 40 mg, 0.072 mmol, 0.26 equiv.), and toluene
(11 mL) under argon. The flask was immersed in an oil bath at
100 °C with stirring until the starting material had completely dis-
appeared as judged by TLC analysis. The solution was then allowed
to cool to room temperature, was diluted with dichloromethane
(100 mL), filtered through Celite, and concentrated. The crude
product was then purified further by column chromatography
(CH2Cl2/MeOH, 45:1) on silica gel to give 10 (74 mg, 0.14 mmol,
1
50%) as a dark-red viscous oil. H NMR (400 MHz, CDCl3): δ =
1.09 (s, 6 H), 2.39 (s, 2 H), 2.52 (s, 3 H), 2.78 (s, 2 H), 2.94 (t, J =
5.47 Hz, 2 H), 3.30 (d, J = 4.81 Hz, 2 H), 3.91 (s, 4 H), 6.09 (br. s,
1 H), 6.69 (dd, J = 8.29, 1.74 Hz, 2 H), 6.74 (d, J = 1.56 Hz, 1 H),
7.16 (dd, J = 6.93, 5.64 Hz, 1 H), 7.48 (d, J = 8.28 Hz, 1 H), 7.58
(d, J = 7.82 Hz, 2 H), 7.69 (dt, J = 7.64, 1.66 Hz, 2 H), 8.56 (d, J =
4.31 Hz) ppm. 13C-Apt (100 MHz, CDCl3): δ = 13.37, 28.37, 29.65,
35.83, 37.64, 40.33, 51.53, 52.30, 60.04, 94.81, 105.60, 110.42,
117.45, 117.51, 122.25 (2 C), 123.26 (2 C), 133.84, 136.85 (2 C),
143.41, 149.04, 149.09 (2 C), 150.28, 151.26, 158.69, 191.23 ppm.
HRMS (ESI): calcd. for C31H34N7O 520.2825; found 520.2826.
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-4-(3,6,6-trimethyl-
4-oxo-4,5,6,7-tetrahydroindazol-1-yl)benzamide (ZnABA, 2): KOH
(22 mg, 0.39 mmol, 5.65 equiv.) and compound 10 (36 mg,
0.069 mmol, 1 equiv.) were added to a solution of EtOH and
DMSO (4:1, 1.12 mL). The reaction mixture was stirred in a 50 °C
oil bath, and H2O2 (30%, 0.11 mL) was slowly added dropwise
through a syringe over 0.5 h. Then the resulting solution was stirred
for another 1.5 h until the disappearance of benzonitrile, as shown
by TLC. After removal of the solvent by rotary evaporation, the
mixture was diluted with dichloromethane (150 mL), washed with
saturated sodium chloride solution (3ϫ 30 mL), dried with magne-
sium sulfate, filtered, and concentrated. The mixture was purified
by column chromatography (CH2Cl2/MeOH, 20:1) to give ZnABA
(32 mg, 0.060 mmol, 87 %) as a colorless solid. 1H NMR
(400 MHz, CDCl3): δ = 1.09 (s, 6 H), 2.39 (s, 2 H), 2.54 (s, 3 H),
2.77 (s, 2 H), 2.91 (t, J = 6.04 Hz, 2 H), 3.32 (t, J = 6.08 Hz, 2 H),
3.90 (s, 4 H), 6.63 (dd, J = 8.40, 1.84 Hz, 1 H), 6.71 (d, J = 1.72 Hz,
1 H), 7.12 (t, J = 6.06 Hz, 1 H), 7.49 (d, J = 8.40 Hz, 1 H), 7.65
(t, J = 7.62 Hz, 2 H), 7.73 (d, J = 7.80, 2 H), 8.48 (d, J = 4.12 Hz,
2 H) ppm. 13C-Apt (100 MHz, CDCl3): δ = 13.39, 28.38 (2 C),
35.77, 37.50, 40.42, 52.36, 52.53, 53.38, 60.35, 106.38, 109.07,
112.95, 117.14, 122.04 (2 C), 123.24 (2 C), 129.48, 136.59 (2 C),
142.57, 148.81 (2 C), 149.03, 149.88, 150.64, 159.14, 171.17,
193.39 ppm. HRMS (ESI): calcd. for C31H36N7O2 538.2930; found
538.2924.
Experimental Section
General: All chemicals were purchased as reagent grade and used
without further purification. Buchwald–Hartwig cross-coupling re-
actions were performed in flame-dried glassware under argon. Tol-
uene was distilled from calcium hydride. The reactions were moni-
tored by analytical thin-layer chromatography (TLC) on silica gel
F254 glass plates and visualized under UV light (254 and 365 nm)
and/or by staining with ninhydrin. Flash column chromatography
was performed on silica gel (200–300 mesh). 1H NMR spectra were
recorded with a Bruker Avance III 400 MHz NMR spectrometer
at 20 °C. Chemical shifts (in ppm) were determined relative to tet-
ramethylsilane (δ = 0 ppm) in deuteriated solvents. Coupling con-
stants in Hz were measured from the one-dimensional spectra. 13
C
NMR or 13C attached-proton-test (13C-Apt) spectra were recorded
with the 400 MHz NMR spectrometer (100 MHz) and calibrated
with CDCl3 (δ = 77.23 ppm). High-resolution mass spectra were
recorded with a Waters LCT Premier XE mass spectrometer. UV
absorption and emission spectra were recorded with a GBC Cintra
10e UV/Vis spectrophotometer and a Varian Cary Eclipse spectro-
fluorimeter, respectively, in a quartz cell with a 1 cm path length.
2-Bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)-
benzonitrile (8): Compound 7[25] (107 mg, 0.60 mmol, 1.20 equiv.)
was dissolved in DMF, and 2-bromo-4-fluorobenzonitrile (6;
100 mg, 0.5 mmol, 1.00 equiv.) and K2CO3 (118 mg, 0.86 mmol,
1.72 equiv.) were added. The reaction mixture was stirred at room
temperature for 30 min and then heated in a 40 °C oil bath until
the starting material had been completely consumed as detected by
TLC. The solution was then allowed to cool to room temperature,
and the DMF was evaporated in vacuo to leave a yellowish oil.
The crude oil was then diluted with dichloromethane (150 mL),
4-Bromo-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)-
benzonitrile (12): Compound 7 (214 mg, 1.2 mmol, 1.2 equiv.) was
dissolved in DMF, and 4-bromo-2-fluorobenzonitrile (11; 200 mg,
1.0 mmol, 1.0 equiv.) and K2CO3 (236 mg, 1.7 mmol, 1.7 equiv.)
were added. The reaction mixture was stirred at room temperature
until the starting material had been completely consumed as judged
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by TLC. After removal of DMF by rotary evaporation in vacuo,
the mixture was then diluted with dichloromethane (150 mL),
washed with saturated sodium chloride solution (3ϫ 30 mL), and
dried with magnesium sulfate. After removal of the solvent, the
mixture was purified by column chromatography (hexanes/EtOAc,
4:1) and then recrystallized (CH2Cl2/hexanes) to give 12 (330 mg,
Schlenk flask was charged with compound 15 (70 mg, 0.13 mmol,
1.0 equiv.), BPEA (50 mg, 0.21 mmol, 1.6 equiv.), sodium tert-bu-
toxide (35.7 mg, 0.37 mmol, 2.8 equiv.), palladium chloride (9 mg,
0.05 mmol, 0.38 equiv.), DPPF (20 mg, 0.036 mmol, 0.27 equiv.),
and toluene (5 mL) under argon. The flask was immersed in an oil
bath at 100 °C with stirring until the starting material had been
0.92 mmol, 92%) as a colorless solid. 1H NMR (400 MHz, CDCl3): completely consumed as judged by TLC. The solution was then
δ = 1.13 (s, 6 H), 2.42 (s, 2 H), 2.55 (s, 3 H), 2.70 (s, 2 H), 7.67–
allowed to cool to room temperature, taken up in dichloromethane
(100 mL), filtered, and concentrated. The crude product was then
7.75 (m, 3 H) ppm. 13C-Apt (100 MHz, CDCl3): δ = 13.32, 28.31,
36.09, 52.4, 109.07, 115.30, 117.30, 128.54, 131.32, 132.79, 134.58, purified further by column chromatography [CH2Cl2/MeOH, 40:1,
141.36, 151.26, 193.12 ppm. HRMS (ESI): calcd. for C17H17BrN3O
358.0555; found 358.0558.
containing 0.1% Et3N (v/v)] on silica gel to give 16 (40 mg,
0.057 mmol, 44%) as a viscous oil. H NMR (400 MHz, CDCl3):
1
δ = 1.13 (s, 12 H), 2.41 (s, 4 H), 2.57 (s, 6 H), 2.74 (s, 4 H), 2.98
(s, 2 H), 3.29 (s, 2 H), 3.97 (s, 4 H), 6.75 (s, 2 H), 7.22 (br. s, 2 H),
7.34 (d, J = 6.16 Hz, 2 H), 7.67 (t, J = 6.84 Hz, 2 H), 8.58 (d, J =
3.90 Hz, 2 H) ppm. Then, by using a procedure similar to that
described for the preparation of ZnABA, compound 15 was hy-
drated by KOH catalyzed by H2O2 to give 4 in 83% yield as a
colorless solid. 1H NMR (400 MHz, CDCl3): δ = 1.08 (s, 12 H),
2.36 (s, 4 H), 2.53 (s, 6 H), 2.57 (s, 4 H), 2.95 (s, 2 H), 3.24 (s, 2
H), 3.94 (s, 4 H), 6.65 (s, 2 H), 7.20 (s, 2 H), 7.37 (s, 2 H), 7.66 (s,
2 H), 8.56 (d, J = 3.90 Hz, 2 H) ppm. 13C-Apt (100 MHz, CDCl3):
δ = 13.45, 28.24, 29.69, 35.52, 35.80, 40.89, 51.70, 52.52, 59.34,
112.18, 116.18, 122.52, 123.54, 136.92, 138.16, 148.94, 149.82,
150.60, 151.79, 165.00, 193.42 ppm. HRMS (ESI): calcd. for
C41H48N9O3 714.3880; found 714.3889.
4-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-2-(3,6,6-trimethyl-
4-oxo-4,5,6,7-tetrahydroindazol-1-yl)benzamide (3): A Schlenk flask
was charged with compound 12 (70 mg, 0.20 mmol, 1.00 equiv.),
BPEA (70 mg, 0.29 mmol, 1.48 equiv.), sodium tert-butoxide
(36 mg, 0.37 mmol, 1.85 equiv.), palladium chloride (19 mg,
0.11 mmol, 0.56 equiv.), DPPF (42 mg, 0.076 mmol, 0.38 equiv.),
and toluene (5 mL) under argon. The flask was immersed in an oil
bath at 100 °C with stirring until the starting material had been
completely consumed as determined by TLC. The solution was
then allowed to cool to room temperature, taken up in dichloro-
methane (100 mL), filtered, and concentrated. The crude product
was then purified further by column chromatography (CH2Cl2/
MeOH, 40:1) on silica gel to give 13 (79 mg, 0.15 mmol, 78%) as
1
a viscous oil. H NMR (400 MHz, CDCl3): δ = 1.11 (s, 6 H), 2.40
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)benzamide (5):
A
(s, 2 H), 2.55 (s, 3 H), 2.70 (s, 2 H), 2.97 (s, 2 H), 3.23 (s, 2 H),
3.96 (s, 4 H), 6.58 (s, 1 H), 6.71 (d, J = 8.60 Hz, 1 H), 7.21 (br. s,
2 H), 7.38 (br. s, 2 H), 7.48 (d, J = 8.65 Hz, 2 H), 7.66 (br. s, 2 H),
8.60 (d, J = 4.43 Hz, 2 H) ppm. Then, by using the same procedure
as that described for the preparation of ZnABA, compound 13 was
hydrated with KOH catalyzed by H2O2 to give 3 in 90% yield as a
colorless solid. 1H NMR (400 MHz, CDCl3): δ = 1.06 (s, 6 H), 2.36
(s, 2 H), 2.43 (s, 2 H), 2.55 (s, 3 H), 2.94 (s, 2 H), 3.22 (s, 2 H),
3.95 (s, 4 H), 6.38 (s, 1 H), 6.75 (dd, J = 8.73, 2.28 Hz, 1 H), 7.18–
7.21 (m, 2 H), 7.39 (br. s, 2 H), 7.63–7.67 (m, 2 H), 7.81 (d, J =
8.66 Hz, 1 H), 8.58 (d, J = 4.60 Hz, 2 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 13.46, 28.21, 35.38, 35.68, 40.93, 51.91,
52.54, 59.85, 110.33, 113.63, 116.36, 118.90, 122.29, 123.24, 132.37,
136.61, 137.00, 149.15, 150.19, 151.86, 152.32, 158.74, 167.79,
193.40 ppm. HRMS (ESI): calcd. for C31H36N7O2 538.2930; found
538.2933.
Schlenk flask was charged with compound 17 (60 mg, 0.33 mmol,
1.14 equiv.), BPEA (71 mg, 0.29 mmol, 1.00 equiv.), sodium tert-
butoxide (70 mg, 0.73 mmol, 2.52 equiv.), palladium chloride
(19 mg, 0.11 mmol, 0.37 equiv.), DPPF (42 mg, 0.076 mmol,
0.26 equiv.), and toluene (13 mL) under argon. The flask was im-
mersed in an oil bath at 100 °C with stirring until the starting mate-
rial had been completely consumed as judged by TLC. The solution
was then allowed to cool to room temperature, taken up in dichlo-
romethane (100 mL), filtered, and concentrated. The crude product
was purified further by column chromatography (CH2Cl2/MeOH,
45:1) on silica gel to give 18 (54 mg, 0.17 mmol, 59%) as a viscous
1
oil. H NMR (400 MHz, CDCl3): δ = 2.95 (s, 2 H), 3.26 (s, 2 H),
3.95 (s, 4 H), 6.54 (d, J = 8.47 Hz, 1 H), 6.63 (t, J = 7.54 Hz, 1 H),
7.18 (t, J = 5.94 Hz, 2 H), 7.30–7.39 (m, 2 H), 7.65–7.75 (m, 4 H),
8.55 (d, J = 4.86 Hz, 2 H) ppm. Then, by using a procedure similar
to that described for the preparation of ZnABA, compound 18 was
hydrated by KOH catalyzed by H2O2 to give 5 in 83% yield as a
colorless solid. 1H NMR (400 MHz, CDCl3): δ = 2.92 (s, 2 H), 3.32
(s, 2 H), 3.95 (s, 4 H), 6.55–6.61 (m, 2 H), 7.16 (br. s, 2 H), 7.39
(d, J = 7.37 Hz, 2 H), 7.69 (t, J = 7.55 Hz, 2 H), 7.79 (d, J =
7.81 Hz, 2 H), 8.51 (d, J = 4.22 Hz, 2 H) ppm. 13C-Apt (100 MHz,
CDCl3/CD3OD): δ = 40.08, 52.74, 60.16, 111.69, 113.68, 114.61,
122.28, 123.34, 128.47, 133.31, 137.14, 148.31, 149.54, 159.10,
172.71 ppm. HRMS (ESI): calcd. for C23H26N3O 362.1981; found
362.1987.
4-Bromo-2,6-bis(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-
yl)benzonitrile (15): Compound 7 (180 mg, 1.01 mmol, 2.2 equiv.)
was dissolved in DMF, and 4-bromo-2,6-difluorobenzonitrile (14;
100 mg, 0.46 mmol, 1.0 equiv.) and K2CO3 (180 mg, 1.30 mmol,
2.8 equiv.) were added. The reaction mixture was stirred at room
temperature until the starting material had been completely con-
sumed as judged by TLC. After removal of DMF by rotary evapo-
ration in vacuo, the mixture was then diluted with dichloromethane
(150 mL), washed with saturated sodium chloride solution (3 ϫ
30 mL), dried with magnesium sulfate, and filtered. After removal
of the solvent, the mixture was purified by column chromatography
(hexanes/EtOAc, 3.5:1) and then recrystallized (CH2Cl2/hexanes)
to give 15 (176 mg, 0.33 mmol, 72%) as a colorless solid. 1H NMR
(400 MHz, CDCl3): δ = 1.14 (s, 12 H), 2.43 (s, 4 H), 2.57 (s, 6 H),
2.74 (s, 4 H), 7.82 (s, 2 H) ppm. 13C NMR (100 MHz, CDCl3): δ =
13.39, 14.13, 22.65, 28.32, 31.58, 35.07, 36.18, 52.34, 60.39, 107.25,
112.55, 117.61, 128.75, 131.34, 142.43, 151.52, 151.78, 193.01 ppm.
HRMS (ESI): calcd. for C27H29BrN5O2 534.1505; found 534.1503.
Spectroscopic Materials and Methods: Stock solutions (0.001 m) of
zinc perchlorate were prepared in HEPES buffer (25 mm HEPES,
0.1 m NaClO4, pH = 7.4, I = 0.1). Stock solutions (0.001 m) of 1a,
ZnABA, 3, 4, and 5 were prepared in EtOH. All the fluorescence
spectra were also measured in HEPES buffer [25 mm HEPES, 0.1 m
NaClO4, pH = 7.4, I = 0.1, 1% (v/v) EtOH], and the excitation
wavelength was 260 nm with excitation and emission slit widths of
5 nm at room temperature.
Supporting Information (see also the footnote on the first page of
this article): Figures S1–S7 and the 1H and 13C NMR spectra of
new compounds.
4-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-2,6-bis(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide (4): A
4614
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 4609–4615

Fluorescent Sensors for Zinc Ion Derived from Aminobenzamide
Am. Chem. Soc. 2003, 125, 10591–10597; c) M. Royzen, A.
Durandin, V. G. Young Jr., N. E. Geacintov, J. W. Canary, J.
Am. Chem. Soc. 2006, 128, 3854–3855; d) Y. Liu, N. Zhang, Y.
Chen, L. H. Wang, Org. Lett. 2007, 9, 315–318; e) X. Y. Chen,
J. Shi, Y. M. Li, F. L. Wang, X. Wu, Q. X. Guo, L. Liu, Org.
Lett. 2009, 11, 4426–4429.
Acknowledgments
This project was financially supported in part by the National Nat-
ural Science Foundation of China (20772013), the Fundamental
Research Funds for the Central Universities and the Beijing
Municipal Commission of Education. The authors are grateful to
Prof. Wei-jun Jin for helpful suggestions on the fluorescence spectra
and Prof. Guo-fu Zi for helpful discussions on the X-ray crystal-
lography.
[14] a) T. Hirano, K. Kikuchi, Y. Urano, T. Higuchi, T. Nagano, J.
Am. Chem. Soc. 2000, 122, 12399–12400; b) T. Hirano, K. Ki-
kuchi, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2002, 124,
6555–6562; c) K. R. Gee, Z. L. Zhou, W. J. Qian, R. Kennedy,
J. Am. Chem. Soc. 2002, 124, 776–778; d) S. C. Burdette, C. J.
Frederickson, W. M. Bu, S. J. Lippard, J. Am. Chem. Soc. 2003,
125, 1778–1787.
[1] B. L. Vallee, K. H. Falchuk, Physiol. Rev. 1993, 73, 79–118.
[2] C. J. Frederickson, Int. Rev. Neurobiol. 1989, 31, 145–238.
[3] P. D. Zalewski, S. H. Millard, I. J. Forbes, O. Kapaniris, A. Sla-
votinek, W. H. Betts, A. D. Ward, S. F. Lincoln, I. Mahadevan,
J. Histochem. Cytochem. 1994, 42, 877–884.
[4] P. D. Zalewski, X. Jian, L. L. L. Soon, W. G. Breed, R. F. Sea-
mark, S. F. Lincoln, A. D. Ward, F. Z. Sun, Reprod. Fertil. Dev.
1996, 8, 1097–1105.
[15] a) A. E. Lee, M. R. Grace, A. G. Meyer, K. L. Tuck, Tetrahe-
dron Lett. 2010, 51, 1161–1165; b) E. U. Akkaya, M. E. Hus-
ton, A. W. Czarnik, J. Am. Chem. Soc. 1990, 112, 3590–3593.
[16] W. Jiang, Q. Fu, H. Fan, W. Wang, Chem. Commun. 2008, 259–
261.
[17] S. Banthia, A. Samanta, Eur. J. Org. Chem. 2005, 4967–4970.
[18] K. Kiyose, H. Kojima, Y. Urano, T. Nagano, J. Am. Chem.
Soc. 2006, 128, 6548–6549.
[5] J. J. R. F. de Silva, R. J. P. Williams in The Biological Chemistry
of Elements: The Inorganic Chemistry of Life, Oxford Univer-
sity Press, New York, 2001.
[6] a) A. I. Bush, Trends Neurosci. 2003, 26, 207–214; b) A. B.
Chausmer, J. Am. Coll. Nutr. 1998, 17, 109–115; c) D. W. Choi,
J. Y. Koh, Annu. Rev. Neurosci. 1998, 21, 347–375; d) A. S. Pra-
sad, “The role of zinc in brain and nerve functions” in Metals
and Oxidative Damage in Neurological Disorders (Ed.: J. R.
Connor), Plenum Press, New York, 1997.
[19] a) K. H. Huang, J. M. Veal, R. P. Fadden, J. W. Rice, J. Eaves,
J. P. Strachan, A. F. Barabasz, B. E. Foley, T. E. Barta, W. Ma,
M. A. Silinski, M. Hu, J. M. Partridge, A. Scott, L. G. DuBois,
T. Freed, P. M. Steed, A. J. Ommen, E. D. Smith, P. F. Hughes,
A. R. Woodward, G. J. Hanson, W. S. McCall, C. J. Mark-
worth, L. Hinkley, M. Jenks, L. Geng, M. Lewis, J. Otto, B.
Pronk, K. Verleysen, S. E. Hall, J. Med. Chem. 2009, 52, 4288–
4305; b) Y. Okawa, T. Hideshima, P. Steed, S. Vallet, S. Hall,
K. Huang, J. Rice, A. Barabasz, B. Foley, H. Ikeda, N. Raje,
T. Kiziltepe, H. Yasui, S. Enatsu, K. C. Anderson, Blood 2009,
113, 846–855.
[7] C. J. Frederickson, E. J. Kasarskis, D. Ringo, R. E. Freder-
ickson, J. Neurosci. Methods 1987, 20, 91–103.
[8] a) K. Kikuchi, K. Komatsu, T. Nagano, Curr. Opin. Chem.
Biol. 2004, 8, 182–191; b) T. Terai, T. Nagano, Curr. Opin.
Chem. Biol. 2008, 12, 515–521; c) E. L. Que, D. W. Domaille,
C. J. Chang, Chem. Rev. 2008, 108, 1517–1549.
[9] a) C. C. Woodroofe, S. J. Lippard, J. Am. Chem. Soc. 2003,
125, 11458–11459; b) C. J. Chang, J. Jaworski, E. M. Nolan,
M. Sheng, S. J. Lippard, Proc. Natl. Acad. Sci. USA 2004, 101,
1129–1134.
[10] a) T. Hirano, K. Kikuchi, Y. Urano, T. Higuchi, T. Nagano, J.
Am. Chem. Soc. 2000, 122, 12399–12400; b) T. Hirano, K. Ki-
kuchi, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2002, 124,
6555–6562; c) S. Maruyama, K. Kikuchi, T. Hirano, Y. Urano,
T. Nagano, J. Am. Chem. Soc. 2002, 124, 10650–10651.
[11] a) K. Hanaoka, Y. Muramatsu, Y. Urano, T. Terai, T. Nagano,
Chem. Eur. J. 2010, 16, 568–572; b) F. Qian, C. Zhang, Y.
Zhang, W. He, X. Gao, P. Hu, Z. Guo, J. Am. Chem. Soc. 2009,
131, 1460–1468.
[20] a) Q. Q. Zhai, G. Q. Gong, Y. Luo, Q. D. Wang, M. Xia, G. W.
Xing, Y. C. Li, J. H. Jiang, Z. Liu, Q. Y. Liu, Y. F. Wang, J.
Pharm. Biomed. Anal. 2010, 53, 1048–1052; b) L. Jin, C. L.
Xiao, C. H. Lu, M. Xia, G. W. Xing, S. Xiong, Q. Y. Liu, H.
Liu, Y. C. Li, F. Ge, Q. D. Wang, Q. Y. He, Y. F. Wang, FEBS
Lett. 2009, 583, 1859–1866.
[21] H. Q. Ju, Y. F. Xiang, B. J. Xin, Y. Pei, J. X. Lu, Q. L. Wang,
M. Xia, C. W. Qian, Z. Ren, S. Y. Wang, Y. F. Wang, G. W.
Xing, Bioorg. Med. Chem. Lett. 2011, 21, 1675–1677.
[22] a) J. P. Wolf, S. Wagaw, S. L. Buchwald, J. Am. Chem. Soc.
1996, 118, 7215–7216; b) B. C. Hamann, J. F. Hartwig, J. Am.
Chem. Soc. 1998, 120, 7369–7370.
[23] Y. Sawaki, Y. Ogata, Bull. Chem. Soc. Jpn. 1981, 54, 793–799.
[24] CCDC-816653 (for ZnABA) contains the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
[12] a) S. Aoki, S. Kaido, H. Fujioka, E. Kimura, Inorg. Chem.
2003, 42, 1023–1030; b) E. Kimura, S. Aoki, E. Kikuta, T.
[25] R. M. Claramunt, C. Lopez, C. Perez-medina, E. Pinilla, M. R.
Torres, J. Elguero, Tetrahedron 2006, 62, 11704–11713.
Koike, Proc. Natl. Acad. Sci. USA 2003, 100, 3731–3736; c) [26] C. Incarvito, M. Lam, B. Rhatigan, A. L. Rheingold, C. J. Qin,
N. C. Lim, L. Yao, H. C. Freake, C. Bruckner, Bioorg. Med.
Chem. Lett. 2003, 13, 2251–2254.
[13] a) C. J. Fahrni, T. V. O’Halloran, J. Am. Chem. Soc. 1999, 121,
11448–11458; b) M. D. Shults, D. A. Pearce, B. Imperiali, J.
A. L. Gavrilova, B. Bosnich, J. Chem. Soc., Dalton Trans. 2001,
3478–3488.
Received: March 21, 2011
Published Online: June 27, 2011
Eur. J. Org. Chem. 2011, 4609–4615
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4615